NMR chemical shifts provide important local structural information for proteins. Consistent structure generation from NMR chemical shift data has recently become feasible for proteins with sizes of up to 130 residues, and such structures are of a quality comparable to those obtained with the standard NMR protocol. In collaboration with Dr. David Baker and his group, we have previously developed a chemical-shift-guided approach to successfully and accurately determine structures on the basis of chemical shifts, for systems less than about 130 amino acids. New work focuses on extending this approach to allow incorporation of easily accessible experimental information. This includes development of a program, Promega, to identify Xaa-Pro peptide bond conformation on the basis of backbone chemical shifts and the amino acid sequence. Using a chemical shift database of proteins of known structure together with the PDB-extracted amino acid preference of cis Xaa-Pro peptide bonds, a cis/trans probability score is calculated from the backbone and C-13(beta) chemical shifts of the proline and its neighboring residues. For an arbitrary number of input chemical shifts, which may include Pro-C-13(gamma), Promega calculates the statistical probability that a Xaa-Pro peptide bond is cis. Besides its potential as a validation tool, Promega is particularly useful for studies of larger proteins where Pro-C-13(gamma) assignments can be challenging. We also have developed a nearal-network guided approach to predict chemical shifts for proteins of known structure, a key step in enhancing the database used by the CS-Rosetta program. Proteins with high-sequence identity but very different folds present a special challenge to sequence-based protein structure prediction methods. In particular, a 56-residue three-helical bundle protein (GA(95)) and an alpha/beta-fold protein (GB(95)), which share 95% sequence identity, were targets in the CASP-8 structure prediction contest. With only 12 out of 300 submitted server-CASP8 models for GA(95) exhibiting the correct fold, this protein proved particularly challenging despite its small size. We have demonstrated that the information contained in NMR chemical shifts can readily be exploited by the CS-Rosetta structure prediction program and yields adequate convergence, even when input chemical shifts are limited to just amide H-1(N) and N-15 or H-1(N) and H-1(alpha) values.